From dylanrahe from gmail.com Fri Oct 2 01:27:51 2009 From: dylanrahe from gmail.com (Dylan Rahe) Date: Fri Oct 2 11:09:56 2009 Subject: Short Vector Multiple Cloning Sites/Polylinkers Message-ID: <950A4212-E5BB-449C-85CE-E02B5BA6556E@gmail.com> Hello all, As a tool, I'd like to create a short (well, as short as possible) DNA sequence that would be essentially an MCS/polylinker. Instead of creating it myself, I was wondering if it were actually already created... Does anyone know of a short MCS? The size to beat is Bluescript: 228 bp! Go! Thanks in advance for your post, thanks all! -Dylan Rahe From virashkgupta from gmail.com Fri Oct 2 07:15:50 2009 From: virashkgupta from gmail.com (Virash Gupta) Date: Fri Oct 2 11:10:05 2009 Subject: Methods Digest, Vol 53, Issue 1 In-Reply-To: <200910011704.n91H4XM08079@net.bio.net> References: <200910011704.n91H4XM08079@net.bio.net> Message-ID: I am surprized by what you are observing. Keep one thing in mind. pUC19 transformed pure clones do not differentiate between LB or LB amp. The plasmid does not provide anything for higher growth of the bacterial cells as it will be same in LB or LB-amp. Plasmid allows only selective growth of transformed cells in LB-amp and not the higher growth. You should get same growth rate of pure culture in Lb as well as Lb-amp. On 10/1/09, methods-request@oat.bio.indiana.edu < methods-request@oat.bio.indiana.edu> wrote: > > Send Methods mailing list submissions to > methods@net.bio.net > > To subscribe or unsubscribe via the World Wide Web, visit > http://www.bio.net/biomail/listinfo/methods > or, via email, send a message with subject or body 'help' to > methods-request@net.bio.net > > You can reach the person managing the list at > methods-owner@net.bio.net > > When replying, please edit your Subject line so it is more specific > than "Re: Contents of Methods digest..." > > > Today's Topics: > > 1. (no subject) (Sissi zhang) > > > ---------------------------------------------------------------------- > > Message: 1 > Date: Mon, 2 Mar 2009 17:44:03 -0800 > From: Sissi zhang > Subject: (no subject) > To: Biosci Bionet news group > Message-ID: > Content-Type: text/plain; charset=3D"windows-1256" > > > > > > > > > > Kyle > > Thank you for the advice. I am still trying to figure out why my culture = is > not growing well. > Now > I just test the starter culture since it did not grow to the expected > cell densities in the first place. The OD600=3D0.75 after 20 hr > incubation at 37C at 300 rpm. The culture was half transparent after 20 > hrs growth. > > Here was the procedure: > The colony was picked from fresh agar plate. > The single colony (around 1.2 mm in diameter) was inoculated to 2.5 mL LB > broth (with or without Amp) > After 20 hrs growth, both tubes gave me the same cell density, OD600=3D0.= 75 > (with or without 100 ug/mL Amp). > I checked the pH of LB broth and adjust it to pH7.5 before autoclave. > > I > really do not know what the trouble is. Transformed DH5 +pUC19 should > grow very fast. I just placed an order for liquid LB broth from > different vendor (not dehydrated LB), in case the problem lies in the > water I used to make LB broth or something. Could it be contamination > issue? But I don't see how. > > The super broth formula you used seems very good. I will try it if the ne= w > LB broth I just ordered will not work. Thanks, Sissi > > _________________________________________________________________ > Windows Live=99 Contacts: Organize your contact list. > > http://windowslive.com/connect/post/marcusatmicrosoft.spaces.live.com-Blo= g-cns!503D1D86EBB2B53C!2285.entry?ocid=3DTXT_TAGLM_WL_UGC_Contacts_032009 > > ------------------------------ > > _______________________________________________ > Methods mailing list > Methods@net.bio.net > http://www.bio.net/biomail/listinfo/methods > > End of Methods Digest, Vol 53, Issue 1 > ************************************** > --=20 Dr V K Gupta Sr Microbiologist (Molecular Biology) Insect Molecular Biology Lab Department of Entomology Punjab Agricultural University Ludhiana (Pb)-141004- India M: 09815963210 From monikagicts1 from gmail.com Sat Oct 3 06:29:03 2009 From: monikagicts1 from gmail.com (ms) Date: Sat Oct 3 12:09:53 2009 Subject: problem with competent cells Message-ID: <85fb6abd-aaef-48bf-aa9e-ba3cf2fe8f14@u36g2000prn.googlegroups.com> hi all, i want to share one problem regarding DH5 alpha ultra com cells preparation using inoue method. i am getting contamination as i am not sure whether it is contamination exactly i donot know but i am getting good efficiency. everything i am doing in sterile environment. all buffers i prepared in hood and filtered through .22 micron filter but i donot know what is the problem. so, please suggest me what is the problem. ms From rahimpour_a from yahoo.com Sat Oct 3 07:30:12 2009 From: rahimpour_a from yahoo.com (Azam Rahimpour) Date: Sat Oct 3 12:09:59 2009 Subject: fusion protein construction Message-ID: <782308.72937.qm@web52705.mail.re2.yahoo.com> Hi I am going to construct a gusion protein between an enzyme (alkaline phosphatase) and a neomycine resitance gene for expression in mammalian cells, I study in some articles that in some cases a linker sequence is added between two proteins,?please let me know if: ? 1. when create a fusion protein what is the risk that individual proteins loss their activity ? 2. is adding a linker necessary and what kind of linker works best ? regards azam ? ? ? ? From cathalgarvey from gmail.com Sat Oct 3 18:01:53 2009 From: cathalgarvey from gmail.com (Cathal Garvey) Date: Sat Oct 3 19:45:36 2009 Subject: fusion protein construction In-Reply-To: <782308.72937.qm@web52705.mail.re2.yahoo.com> References: <782308.72937.qm@web52705.mail.re2.yahoo.com> Message-ID: <468b1a400910031601m65a3937ai2ade6bcac30c4482@mail.gmail.com> Hi Azam, If you just want to have both genes active in the transformed cells, then it is not necessary to fuse them. Doing so might inactivate the function of one or both. If, on the other hand, you aim for them to perform a function together, then fusion may indeed be a good way to do it. Fused proteins have been observed to provide far greater activity than the separate proteins might if co-expressed, assuming they work upon the same substrate. The linker region should be at least 8-10 amino acids long, I think, assuming the C-terminal end of the first protein and the N-terminal end of the second protein are both exposed on the outer surface of the proteins (if in doubt, they probably are). Try to pick inert amino acids that don't force structural changes due to their shape. A pair of simple uncharged amino acids repeated 4-5 times should do, I think. I hope this helped! Also, don't take it as authoritative. I've only read a few papers on the matter. -Cathal 2009/10/3 Azam Rahimpour > Hi > I am going to construct a gusion protein between an enzyme (alkaline > phosphatase) and a neomycine resitance gene for expression in mammalian > cells, I study in some articles that in some cases a linker sequence is > added between two proteins, please let me know if: > > 1. when create a fusion protein what is the risk that individual proteins > loss their activity > > 2. is adding a linker necessary and what kind of linker works best > > regards > azam > > > > > > > > _______________________________________________ > Methods mailing list > Methods@net.bio.net > http://www.bio.net/biomail/listinfo/methods > -- letters.cunningprojects.com twitter.com/onetruecathal Kiva.org - Loans That Change Lives From bzahedi from bccrc.ca Mon Oct 5 00:32:55 2009 From: bzahedi from bccrc.ca (Bari Zahedi) Date: Mon Oct 5 12:37:57 2009 Subject: problem with competent cells In-Reply-To: <85fb6abd-aaef-48bf-aa9e-ba3cf2fe8f14@u36g2000prn.googlegroups.com> References: <85fb6abd-aaef-48bf-aa9e-ba3cf2fe8f14@u36g2000prn.googlegroups.com> Message-ID: About a year ago, my lab and another was getting contamination. We had someone look at what was growing in our cultures and they suggested it was yeast. We narrowed down the source to our 0.25micron columns that we were using to filter our media and reagents. I brought this up with the company and they did alot of testing and decided that it was not yeast and thus did not believe me that what ever it was was coming from their columns. We stopped ordering columns from them and have never had the problem since. I thought i would pass that along. But comtamination could be coming from anywhere really. Here are somethings to try to narrow it down incase you have not done so already. When you say contamination do you mean fungus growing on your plates or another plasmid is coming out of your plasmid preps? If it is something like fungus, then my suggestion is to 1)go to another lab and borrow reagents from them to grow up your DH5a. 2) If it is coming from your reagents, then you will have to figure it out through elimination. My suggestion is to try test a bunch at one time- this kinda saves time. You can also try testing your reagents with mock transformation with no cells. It may be informative. And perhaps if none of this works see if it is the columns! If the contamination is a plasmid, 3)then streak a bit of your cells on your agar + antibiotic plates and see if anything grows. If it does, plasmid prep it and see if it is the same as the contamination you have been getting. 4) Wash your pipettes well, inside and out and use filtered tips. I have been able to eliminate contamination in this way from sets of pipettes in our lab. These are just a few to try. good luck. ________________________________________ From: methods-bounces@oat.bio.indiana.edu [methods-bounces@oat.bio.indiana.edu] On Behalf Of ms [monikagicts1@gmail.com] Sent: October 3, 2009 4:29 AM To: methods@magpie.bio.indiana.edu Subject: problem with competent cells hi all, i want to share one problem regarding DH5 alpha ultra com cells preparation using inoue method. i am getting contamination as i am not sure whether it is contamination exactly i donot know but i am getting good efficiency. everything i am doing in sterile environment. all buffers i prepared in hood and filtered through .22 micron filter but i donot know what is the problem. so, please suggest me what is the problem. ms _______________________________________________ Methods mailing list Methods@net.bio.net http://www.bio.net/biomail/listinfo/methods From luisnevescunha from gmail.com Mon Oct 5 01:48:17 2009 From: luisnevescunha from gmail.com (luiscunhamx) Date: Mon Oct 5 12:38:04 2009 Subject: Mitochondrial isolation for mt DNA extraction from Earthworms Message-ID: <1599165b-fdb2-4314-baef-3ed119008221@f20g2000prn.googlegroups.com> Dear collegues I wonder if anyone ever tried to isolate mitochondrias from earthworms for posterior mtDNA extraction, could anyone send me a good protocol? I am having a lot of trouble doing that, probably due to soil contamination in the digestive tract, even after depuration and I am getting very low yield of mtDNA. Any help?? I am working with Pontoscolex corethrurus, my model organism :). Best regards From monikagicts1 from gmail.com Wed Oct 7 04:50:14 2009 From: monikagicts1 from gmail.com (monika singh) Date: Wed Oct 7 13:20:46 2009 Subject: hi Message-ID: hi all, i am doing site directed mutagenesis for single amino acid change .i have used both stratagene as well as phusion kit but i didnot get the mutation . i have also found out that the melting temp. of primers which i am using is between 60 to 65 degrees. But according to stratagene primer guidelines it should be greater than or equals to 78 degrres. So, this could be the possibility of not working of my mutation. also,i have used top 10 cells ultra comp cells for transformation. i get the colonies also but it was not positive. so, suggest me regarding com cells, pcr conditions ,dpn1 digestion so that i can get result thanks ms From engelbert_buxbaum from hotmail.com Wed Oct 7 08:08:14 2009 From: engelbert_buxbaum from hotmail.com (Dr Engelbert Buxbaum) Date: Wed Oct 7 13:20:51 2009 Subject: Mitochondrial isolation for mt DNA extraction from Earthworms References: <1599165b-fdb2-4314-baef-3ed119008221@f20g2000prn.googlegroups.com> Message-ID: Am 05.10.2009, 02:48 Uhr, schrieb luiscunhamx : > Dear collegues > I wonder if anyone ever tried to isolate mitochondrias from earthworms > for posterior mtDNA extraction, could anyone send me a good protocol? > I am having a lot of trouble doing that, probably due to soil > contamination in the digestive tract, even after depuration and I am > getting very low yield of mtDNA. Any help?? I am working with > Pontoscolex corethrurus, my model organism :). For histology, it is customary to feed earthworms for two weeks on humid filter paper pieces, which are changed daily. That removes any soil from their digestive tract, which would otherwise nick the microtome knife. From pdeitik from bcm.tmc.edu Wed Oct 7 11:04:11 2009 From: pdeitik from bcm.tmc.edu (Deitiker, Philip R) Date: Wed Oct 7 13:21:02 2009 Subject: Mitochondrial isolation for mt DNA extraction from Earthworms In-Reply-To: <1599165b-fdb2-4314-baef-3ed119008221@f20g2000prn.googlegroups.com> References: <1599165b-fdb2-4314-baef-3ed119008221@f20g2000prn.googlegroups.com> Message-ID: I am not really sure whether this reply will help or not, I used to work on C. Elegans. As part of that work one of the topics we touched upon was worm metabolism. When certain deep soil worms (worms that live in deeper anoxic layers of the soil or in wetlands) are exposed to low oxygen environments they tend to shut down classic oxidative catabolism and revert to a low oxygen using pathway (mammals don't have this pathway). Mitochondria respond to signals and may decrease in number when challenged. One possible way to increase mitochondrial DNA is to increase mitochondria by feeding these worms a preferred substrate in a better oxygenated environment. Hope this helps, though doubt it will. BTW, despite the fact that mtDNA has a higher rate of mutations there are some highly conserved regions of mtDNA, I did a study of all the known therian mtDNA sequences and found something >4000 sites that have not evolved in any of the sequenced mtDNA. You should be able to amplify using PCR between these highly conserved regions with a high fidelity polymerase like Fusion, but you will need a clean source of DNA. Since mtDNA is circular you might want to first nick the mtDNA trying different rare cutting endonucleases that cut these conserved sites from known mtDNA sequence within the local clade. At 17,000 nucleotide you should be able to amplify within 8.5 minutes the entire mitogenome. IOW, you should be able to isolate DNA from single cells, or washed egg cells. Oh and one other thing, since mtDNA is circular (I assume it is circular in this species) it will be more difficult to isolate than autosomal DNA strands, it will none the less precipitate, if you are using a DNA isolation kit, cool the isopropanol mixture down to 4'C and let the DNA settle down for a few hours before centrifugation, and be careful the DNA may not be in a concise pellet, but spread around the bottom of the tube. Sonication, if used in the preparation will produce random breaks in the DNA and make it linear. -----Original Message----- From: methods-bounces@oat.bio.indiana.edu [mailto:methods-bounces@oat.bio.indiana.edu] On Behalf Of luiscunhamx Sent: Monday, October 05, 2009 1:48 AM To: methods@magpie.bio.indiana.edu Subject: Mitochondrial isolation for mt DNA extraction from Earthworms Dear collegues I wonder if anyone ever tried to isolate mitochondrias from earthworms for posterior mtDNA extraction, could anyone send me a good protocol? I am having a lot of trouble doing that, probably due to soil contamination in the digestive tract, even after depuration and I am getting very low yield of mtDNA. Any help?? I am working with Pontoscolex corethrurus, my model organism :). Best regards _______________________________________________ Methods mailing list Methods@net.bio.net http://www.bio.net/biomail/listinfo/methods From davidminde from gmail.com Wed Oct 7 14:49:03 2009 From: davidminde from gmail.com (David-Paul Minde) Date: Wed Oct 7 15:44:45 2009 Subject: hi In-Reply-To: References: Message-ID: <123243900910071249v6dc67950h50ae603b732d4ac6@mail.gmail.com> if you can, design primers with silent mutation to check immediately for positives by restriction analysis. Then indeed prolongation of primers can help to get more positives. Also extension of DpnI treatment (or different mutagenesis strategy with exponential amplification as in NEB kit, which however requires more expensive ultra-homogenous primers) to reduce background (also make sure you mix properly before incubation and avoid condensation artefacts). Phusion polymerse (or derivatives of that idea) is better for big amplicons (works sill perfectly for mutations in a 15 kbp plasmid). cheers, David From timepedal from gmail.com Thu Oct 8 13:28:32 2009 From: timepedal from gmail.com (time pedal) Date: Thu Oct 8 14:10:04 2009 Subject: qRT-PCR on very few (10,000 - 20,000 cells) In-Reply-To: References: <200909171707.n8HH7up16704@net.bio.net> Message-ID: <33996c7e0910081128k711c0a41y6eb332026aab470e@mail.gmail.com> Hi everybody, Which qPCR machine is the best choice in your opinions? Need to buy one but not sure what to look at first. Many thanks. T On Thu, Sep 17, 2009 at 2:30 PM, Ann Sutherland wrote: > Hi Adam, >> > > I have routinely done RT-PCR on samples of 100 mouse embryos at the 8-cell > to blastocyst stage (so 800 to 6,000 cells total) with a wide variety of > primers. I think you may not be quantitatively recovering your RNA in step > 2. For large samples, Trizol works really well, but for small samples I > think the percentage lost is too high. Instead of Trizol, I used the > Micro-Fast Track kit from Invitrogen, and later the micro PolyA+ kit from > Ambion to isolate polyA+ RNA from small samples and got great results. I > also routinely use glycogen to co-precipitate with the RNA to get better > yield. > Ann > > ------------------------------ >> >> Hi all, >> >> I am attempting to do qRT-PCR using material from 10,000 - 20,000 cells. >> Here is what I'm doing: >> >> 1) FACS sort 10,000 - 20,000 cells into Trizol LS >> 2) Prepare RNA in Trizol LS per standard protocols >> 3) Make cDNA from the RNA using Invitrogen's SuperScript III First-Strand >> Synthesis System for RT-PCR >> 4) Perform qPCR on that cDNA using Applied Biosystem's RNA-to-Ct kit using >> Sybr green as a detector >> >> I have verified using 100,000 cells that I can amplify my target product. >> The problem is that when I go down to my actual samples of 10,000 cells, >> nothing amplifies, not even my housekeeping gene. I've seen people publish >> qRT-PCR on rare cell populations, but they rarely tell you how many cells >> they actually had to collect to get any usable data. Unfortunately for me, >> those 10,000 cells requires 5-10 transgenic mice and 5 hours of prep time, >> so simply increasing the cell number is probably not viable. >> >> Any suggestions on how to fix this? Do any of you routinely amplify such >> small amounts of RNA for qPCR or qRT-PCR? Do you amplify your RNA first >> using commercially available kits. If so, what kits? >> >> Thanks, >> Adam >> >> >> ------------------------------ >> >> _______________________________________________ >> Methods mailing list >> Methods@net.bio.net >> http://www.bio.net/biomail/listinfo/methods >> >> End of Methods Digest, Vol 52, Issue 11 >> *************************************** >> > > > -- > Ann Sutherland > Associate Professor > Department of Cell Biology > University of Virginia Health System > 1300 Jefferson Park Ave. > Jordan Hall 3-15 > Charlottesville, VA 22908 > > ph: 434-243-6711 > FAX: 434-982-3912 > email: as9n@virginia.edu > > _______________________________________________ > Methods mailing list > Methods@net.bio.net > http://www.bio.net/biomail/listinfo/methods > From gerchman from research.haifa.ac.il Thu Oct 8 13:17:05 2009 From: gerchman from research.haifa.ac.il (Yoram Gerchman) Date: Thu Oct 8 14:10:11 2009 Subject: PhiX174 resistance E. coli strain? Message-ID: <1255025825.4ace2ca1d0178@webmail.haifa.ac.il> Greetings all I am looking for PhiX174 resistance E. coli strain. Does anyone know of such strain? Many thanks Yoram ------------------------------------------------------------------------ This message was sent using IMP, the Webmail Program of Haifa University From rory.obrien from stonebow.otago.ac.nz Thu Oct 8 23:01:20 2009 From: rory.obrien from stonebow.otago.ac.nz (Rory O'Brien) Date: Thu Oct 8 23:10:18 2009 Subject: 3' end labelling with tritiated thymidine... Message-ID: <00c401ca4895$28a35440$79e9fcc0$@obrien@stonebow.otago.ac.nz> Hi Group, I'm looking to try end-tailing an oligonucleotide with terminal transferase and labeling it with tritium in the process; the tritiated thymidine products available from Perkin Elmer come as - NET221H - Deoxythymidine 5`-Triphosphate, [Methyl-3H]-, Tetrasodium Salt, Specific Activity: 10-25Ci (370-925GBq)/mMole, >97%, 1mCi/mL. Packed in ethanol :water (1:1) NET221X - Deoxythymidine 5`-Triphosphate, [Methyl-3H]-, Tetrasodium Salt, Specific Activity: 70-90Ci (2.59-3.33TBq)/mMole, 1mCi/mL. Packed in ethanol :water (1:1) NET221A - Deoxythymidine 5`-Triphosphate, [Methyl-3H]-, Tetrasodium Salt, Specific Activity: 70-90Ci (2.59-3.33TBq)/mMole, 2.5mCi/mL. Packed in 10mM tricine buffer (pH 7.6) Could anyone maybe suggest which thymidine product might be most appropriate to this application? Detection to be via scintillation counter; one for the old-school molecular biologists maybe ;-) Cheers, - Rory From Sharon.Waldrop from utsouthwestern.edu Fri Oct 9 14:56:12 2009 From: Sharon.Waldrop from utsouthwestern.edu (Sharon Waldrop) Date: Fri Oct 9 15:01:25 2009 Subject: IP Question Message-ID: <4ACF4F0C0200003E00050978@swnw126.swmed.edu> Hello everyone, I have some experience with IP but it was a LONG time ago. I know that times have changed. I need help. What kits do you recommend? Any protocols that you use that work for you. Any help and suggestions would be greatly appreciated. Thank you and have a good fay. Shar From rahimpour_a from yahoo.com Fri Oct 9 14:41:00 2009 From: rahimpour_a from yahoo.com (Azam Rahimpour) Date: Fri Oct 9 17:22:52 2009 Subject: rna storage at -20 Message-ID: <107532.37893.qm@web52707.mail.re2.yahoo.com> Hi It is possible to store RNA for short time (1-2 days) at -20 instead of -70? ? regards From hroychow from nmsu.edu Sat Oct 10 11:06:40 2009 From: hroychow from nmsu.edu (Dr. Hiranya S. Roychowdhury) Date: Sat Oct 10 13:28:26 2009 Subject: rna storage at -20 In-Reply-To: <107532.37893.qm@web52707.mail.re2.yahoo.com> References: <107532.37893.qm@web52707.mail.re2.yahoo.com> Message-ID: <1138.71.33.41.235.1255190800.squirrel@webmail.nmsu.edu> Absolutely. I have stored clean RNA prep (properly buffered) without any detectable degradation (as seen via radiolabled probes) for over a month at -20. The keywords are "clean!" and "buffered." > Hi > It is possible to store RNA for short time (1-2 days) at -20 instead of > -70? > ? > regards > > > > _______________________________________________ > Methods mailing list > Methods@net.bio.net > http://www.bio.net/biomail/listinfo/methods > -- Hiranya S. Roychowdhury, Ph.D. Asst. Professor, Health & Public Services Dona Ana Community College New Mexico State University Las Cruces, NM 88003 From itisam.sarangi from gmail.com Sat Oct 10 03:27:18 2009 From: itisam.sarangi from gmail.com (Itisam Sarangi) Date: Sat Oct 10 13:28:33 2009 Subject: Maximun DNA size in miniprep Message-ID: <25de2fb70910100127v6de4fc99nbe955b50caeeccb6@mail.gmail.com> Hi, I am facing a small problem in purifying a plasmid DNA form bactreia of 26kb in size using miniprep express matrix of Mp biomedicals. I think this size of DNA can not be purified by using matrix. Does any one has any ideas waht is the maximum size of plasmid that can be purified thruough matrix. Thanking you Itisam Sarangi From shifalich from rediffmail.com Sat Oct 10 09:56:18 2009 From: shifalich from rediffmail.com (shifali chatrath) Date: Sat Oct 10 13:28:38 2009 Subject: hi Message-ID: <1255125551.S.10372.32403.f4mail-235-241.rediffmail.com.1255186578.62937@webmail.rediffmail.com> Thanks DK! The following info was quite useful for me too. We can't find such details anywhere!!!! Few months back, I was trying to use pfu but my PCRs did n't work, so I gave up after 2-3 trials. Now, I'll using thse tips. Thanks again REgards Shifali On Sat, 10 Oct 2009 03:29:11 +0530 wrote >In article , monika singh wrote: >hi all, > >i am doing site directed mutagenesis for single amino acid change .i have >used both stratagene as well as phusion kit but i didnot get the mutation . >i have also found out that the melting temp. of primers which i am using is >between 60 to 65 degrees. But according to stratagene primer guidelines it >should be greater than or equals to 78 degrres. So, this could be the >possibility of not working of my mutation. also,i have used top 10 cells >ultra comp cells for transformation. i get the colonies also but it was not >positive. so, suggest me regarding com cells, pcr conditions ,dpn1 digestion >so that i can get result Without doing all kind of controls, you can't know where the problem is. The calculation of primers melting temperature is somewhat of a joke as it can vary over 10 degrees in either direction depending on the program you use. Competent cells have nothing to with it as long as you do get colonies. Below is a protocol that I wrote for our lab internal use long ago. It practically never fails. We've done all kind of mutagenesis with it, deletions of >1 kbp and insertions of up to 36 bp. >>>> Although the regular QuickChange works, it does not always work well enough. There are several advantages to the new protocol: - twice less money spent on primers; - twice less polymerase is used; - polymerase with lower error rate is used. Here is a generic protocol followed by notes and comments that might be helpful. Reaction mix (25 ul final) 50 ng template 0.2 uM final single primer 0.2 mM final dNTP mix 2.5 ul 10X Stratagene's PfuUltra II Fusion ("Pfu Fusion" for brevity) buffer 1.0 ul DMSO (or no DMSO) water to 24.5 ml 0.5 ml of Pfu Fusion Cycling conditions 95? 3 min, then 30 cycles: 95? 30 sec 55? 1 min 65? 1 min/kbp template 65? 5 min 4? hold - Dpn I treatment and transformation: Use Fermentas "Quick Digest" DpnI - it is simply a more concentrated version of the enzyme and it costs the same as regular version. Add 10 ul of the amplification reaction to 0.5 ul DpnI, incubate for 2 hours in PCR machine at 37?. During incubation, at least once remove the tube and vortex/spin it again, then continue incubation. - Electroporate using 1-2 ul of the DpnI-treated reaction, immediately add 0.5-1 ml SOC, let cells recover for 45 min at 37?, plate 200 and 20 ml (recovery is not absolutely necessary but it does seem to increase efficiency somewhat). The typical result is several-fold less colonies than in a "standard" QuickChange reaction, and the efficiency in the 50-90% range. I.e., screen 5-6 clones to be sure to get right one no matter what. NOTES AND COMMENTS (in no particular order; I am just mentioning everything that I can think of being worth consideration) 1. Template: Ideally, it should be a fresh, never frozen miniprep. Freeze/thaw produces nicks in the plasmid and nicks are killers to the whole plasmid amplification. It also helps for it to bew "clean" so that UV measures concentration of the plasmid and not just nucleic acids with RNA being a major contaminant. 2. The Pfu Fusion polymerase is a new product that is slightly less expensive than any of the other polymerases we use. It works ~ 4 times faster, makes a bit less errors than Pfu Ultra and its reaction buffer has pH of ~ 10.0. 3. The method requires only one primer - just write down the sequence of the sense strand! It's been found in numerous trials by that desalted primers from IDT work as well as purified ones. 4. Primer design. Keep primer length between 40 and 60 bp. Try not to exceed 60 bp because above that size IDT forces you to order 100 nmol scale with the corresponding price increase. Ideally, you'd want 15-25 bp on either side of the mismatch, approximately equal melting temperature (TM) for both "arms" of the primer (but the "right arm", e.g. 3' part, is more significant because that's where synthesis starts; try to end it with a couple of G/Cs). The overall primer TM (not counting the mismatched pairs) should be in the range of 60-70?C (as calculated by IDT's Oligo Analyzer with default settings). As an example, the most recent primer that worked 100% to delete 6 bases: Left arm 27 bp/51.1?, right arm 19 bp/53.4?, overall 46 bp/63.8? and it worked with 55? annealing. 5. In linear amplification applications (sequencing, QuikChange), the larger number of cycles gives better results. 30X has been tried and works very well. To be sure, 20X also works, although it was not compared to 30X side by side. 6. Amount of template. The stated 50 ng is simply lifted from what we find a near optimum for the standard QuickChanges, 100 ng/50 ul, and was not tested experimentally. In practice, simply using 0.5-2 ul minipreps (depending on culture volume and on plasmid copy number) works fine. In theory, advantages of higher template concentration are: i) more product with less number of cycles, ii) higher chance of priming when annealing is suboptimal. The theoretical disadvantages of having too much of the template are: i) introducing more crap into reaction, some of which might be inhibitory (minipreprs are not clean!), ii) increased background if the Dpn I treatment is incomplete, iii) increase in the misprimed side reactions. There is an optimal balance between all of these factors somewhere... Other things being equal, the best practice is to have as clean template as possible and that means: i) don't be greedy with your minipreps; for high copy number plasmids, 1 ml culture is usually sufficient, 3 ml is a lot and more than 3 ml borders on insanity; ii) use PB solution volume so that it covers all areas of the column that were in contact with cell lysate; iii) use PE solution volume so that it covers all areas of the column that were in contact with PB, and use PE wash TWICE. 7. Annealing temperature. Pfu Fusion seems to be less finicky in terms of annealing conditions. 2-10 degrees below calculated primer TM have been found to work in various cases. 55? should work in the vast majority of cases. 8. DMSO concentration. DMSO is optional as it rarely makes all or none difference. Its main effect on the reaction is two-fold: it decreases melting temperature of DNA duplexes and it reduces primers' secondary structure. The protocol above uses 4% final. You may also try 0 and 6% as a substitute to an annealing temperature variance (make sure to mix DMSO well with other components before adding polymerase). 9. Extension time. Pfu Fusion is quite fast. The default is 60 sec per kbp at 65?C and it definitely works. Tried alternative is 30 sec at 68?C and, for sure, it also works. However, in "QuikChange cloning" trials we found that 65? gives more colonies (perhaps because of the some strand displacement activity at 68?). 10. Other things that may sometimes be beneficial. Increased concentration of the primer two-fold, increased concentration of dNTPs two-fold, an extra 2 mM MgCl2 in the reaction all, in theory, have an effect of increasing product yield with concomitant slight increase in the error rate (not a big deal given the error rate of Pfu Fusion). 11. Just like regular QuickChange, the single stranded QuickChange works for two mutations simultaneously but with reduced efficiency. Make primers so that they have similar TMs and expect 20-30% of the clones to be double mutants. From nick.theodorakis from gmail.com Sat Oct 10 19:41:00 2009 From: nick.theodorakis from gmail.com (Nick Theodorakis) Date: Sun Oct 11 01:23:10 2009 Subject: rna storage at -20 References: <107532.37893.qm@web52707.mail.re2.yahoo.com> Message-ID: On Oct 10, 11:06?am, "Dr. Hiranya S. Roychowdhury" wrote: > Absolutely. ?I have stored clean RNA prep (properly buffered) without any > detectable degradation (as seen via radiolabled probes) for over a month > at -20. ?The keywords are "clean!" and "buffered." > I'll agree with Hiranya, and add that it's preferable if the freezer is a non-defrosting (i.e., not "frost-free") type. Nick -- Nick Theodorakis nick_theodorakis@hotmail.com contact form: http://theodorakis.net/contact.html From itisam.sarangi from gmail.com Mon Oct 12 07:48:21 2009 From: itisam.sarangi from gmail.com (Itisam Sarangi) Date: Mon Oct 12 12:01:11 2009 Subject: Maximun size of DNA obtained thru miniprep matrix Message-ID: <25de2fb70910120548u4a06e676n700ebbdf4ba826ff@mail.gmail.com> Hi all, Thanks DK for the response. this matrix we use to do mini preps in our lab. basically it is a DNA binding matrix originally present in guanidine thiocyante soln. http://www.qbiogene.com/products/dna-rna-purification/miniprep-express.shtml it is this thing, sry cud not get much info abt it online though Just curious to know what is the maximum size of DNA that can be purified thru these matrixes? any inforrmation will be helpful. Thanks Itisam > Message: 5 > Date: Sun, 11 Oct 2009 04:30:41 GMT > From: dk@no.email.thankstospam.net (DK) > Subject: Re: Maximun DNA size in miniprep > To: methods@net.bio.net > Message-ID: > > In article , Itisam > Sarangi wrote: > >Hi, > >I am facing a small problem in purifying a plasmid DNA form bactreia of > 26kb > >in size using miniprep express matrix of Mp biomedicals. > >I think this size of DNA can not be purified by using matrix. > >Does any one has any ideas waht is the maximum size of plasmid that can be > >purified thruough matrix. > > I have no idea what "miniprep express matrix of Mp biomedicals" is. > Do you? > > That said, I've purified ~165 kbp plasmids and genomic DNA > 50 kbp > on Qiagen miniprep columns, eluting with hot TE. No problem. The > recovery is maybe only around 50% but that's plenty enough for > just about anything. Neither is what Qiagen itself says the columns > can handle, by the way. > > DK > > > ------------------------------ > From blackhole from abuse.plus.com Mon Oct 12 09:24:57 2009 From: blackhole from abuse.plus.com (Duncan Clark) Date: Mon Oct 12 12:01:19 2009 Subject: PhiX174 resistance E. coli strain? References: Message-ID: Historians believe that in newspost on Thu, 8 Oct 2009, Yoram Gerchman penned the following literary masterpiece: >I am looking for PhiX174 resistance E. coli strain. Does anyone know of such >strain? The original hosts for phiX174 production were E.coli C derivatives. No idea if it goes into E.coli B or K12 strains. If it's genome has an EcoB or EcoK site in the sequence then that would answer the question. I think a Pubmed search is the best way to go. Duncan -- I love deadlines. I especially like the whooshing noise they make as they go flying by. Duncan Clark GeneSys Ltd. From prae from gmx.net Mon Oct 12 04:24:30 2009 From: prae from gmx.net (Christian Praetorius) Date: Mon Oct 12 12:01:26 2009 Subject: Maximun DNA size in miniprep References: Message-ID: <7jgaugF351488U1@mid.individual.net> dk@no.email.thankstospam.net (DK) wrote: >just about anything. Neither is what Qiagen itself says the columns >can handle, by the way. Of course not. They want to sell more expensive kits, which are specialized for this application. By the way, how did you modify the protocol for the miniprep kit to use it like this? Christian -- X-no-Sig: yes From itisam.sarangi from gmail.com Mon Oct 12 07:48:21 2009 From: itisam.sarangi from gmail.com (Itisam Sarangi) Date: Mon Oct 12 12:01:31 2009 Subject: Maximun size of DNA obtained thru miniprep matrix Message-ID: <25de2fb70910120548u4a06e676n700ebbdf4ba826ff@mail.gmail.com> Hi all, Thanks DK for the response. this matrix we use to do mini preps in our lab. basically it is a DNA binding matrix originally present in guanidine thiocyante soln. http://www.qbiogene.com/products/dna-rna-purification/miniprep-express.shtml it is this thing, sry cud not get much info abt it online though Just curious to know what is the maximum size of DNA that can be purified thru these matrixes? any inforrmation will be helpful. Thanks Itisam > Message: 5 > Date: Sun, 11 Oct 2009 04:30:41 GMT > From: dk@no.email.thankstospam.net (DK) > Subject: Re: Maximun DNA size in miniprep > To: methods@net.bio.net > Message-ID: > > In article , Itisam > Sarangi wrote: > >Hi, > >I am facing a small problem in purifying a plasmid DNA form bactreia of > 26kb > >in size using miniprep express matrix of Mp biomedicals. > >I think this size of DNA can not be purified by using matrix. > >Does any one has any ideas waht is the maximum size of plasmid that can be > >purified thruough matrix. > > I have no idea what "miniprep express matrix of Mp biomedicals" is. > Do you? > > That said, I've purified ~165 kbp plasmids and genomic DNA > 50 kbp > on Qiagen miniprep columns, eluting with hot TE. No problem. The > recovery is maybe only around 50% but that's plenty enough for > just about anything. Neither is what Qiagen itself says the columns > can handle, by the way. > > DK > > > ------------------------------ > From msamet1 from verizon.net Mon Oct 12 08:34:34 2009 From: msamet1 from verizon.net (Marc Samet) Date: Mon Oct 12 12:01:37 2009 Subject: Free siRNA Design software Message-ID: <000001ca4b40$bd10b7a0$373226e0$@net> Might I get a copy of this freeware for siRNA design Many thanks Marc Marc K. Samet, PhD 2214 Windsor Circle Broomall, PA 19008 (610) 353-6750 FAX (501) 635-5353 From gerchman from research.haifa.ac.il Mon Oct 12 12:44:31 2009 From: gerchman from research.haifa.ac.il (Yoram Gerchman) Date: Mon Oct 12 13:34:50 2009 Subject: PhiX174 resistance E. coli strain? In-Reply-To: <200910121703.n9CH3wM16797@net.bio.net> References: <200910121703.n9CH3wM16797@net.bio.net> Message-ID: <1255369471.4ad36affb2cab@webmail.haifa.ac.il> Thanks Dunken. Could you kindly elaborate on the EcoB/EcoK issue? Since the lysis system (E protein) seem to work in K12/coli B whay would not the phage propagate in them? Many thanks Yoram > Date: Mon, 12 Oct 2009 15:24:57 +0100 > From: Duncan Clark > Subject: Re: PhiX174 resistance E. coli strain? > > Historians believe that in newspost > on Thu, 8 Oct 2009, > Yoram Gerchman penned the following > literary masterpiece: > >I am looking for PhiX174 resistance E. coli strain. Does anyone know of such > >strain? > > The original hosts for phiX174 production were E.coli C derivatives. No > idea if it goes into E.coli B or K12 strains. If it's genome has an EcoB > or EcoK site in the sequence then that would answer the question. > > I think a Pubmed search is the best way to go. > > Duncan > -- > I love deadlines. I especially like the whooshing noise they make as > they go flying by. > > Duncan Clark > GeneSys Ltd. ------------------------------------------------------------------------ This message was sent using IMP, the Webmail Program of Haifa University From blackhole from abuse.plus.com Mon Oct 12 11:38:32 2009 From: blackhole from abuse.plus.com (Duncan Clark) Date: Mon Oct 12 13:34:58 2009 Subject: 3' end labelling with tritiated thymidine... References: Message-ID: Historians believe that in newspost on Fri, 9 Oct 2009, Rory O'Brien penned the following literary masterpiece: >Could anyone maybe suggest which thymidine product might be most appropriate >to this application? Detection to be via scintillation counter; one for the >old-school molecular biologists maybe ;-) Highest specific activity would be best so either of the last two. Whether they are the least expensive is another matter. If you are simply using it to check the incorporation even the first one should do. You will just see lower counts. Duncan -- I love deadlines. I especially like the whooshing noise they make as they go flying by. Duncan Clark GeneSys Ltd. From gerchman from research.haifa.ac.il Mon Oct 12 13:22:08 2009 From: gerchman from research.haifa.ac.il (Yoram Gerchman) Date: Mon Oct 12 14:04:29 2009 Subject: PhiX174 resistance E. coli strain? In-Reply-To: <200910121703.n9CH3wM16797@net.bio.net> References: <200910121703.n9CH3wM16797@net.bio.net> Message-ID: <1255371728.4ad373d07832e@webmail.haifa.ac.il> OK, after some digging (OK, a lot of digging) I got it. So EcoB and EcoK are Type I restriction enzymes and will degrade the phiX174 genome. Should have thought about it. Many thanks again, always nice to learn something new! Yoram ------------------------------------------------------------------------ This message was sent using IMP, the Webmail Program of Haifa University From allan.jones from gmx.de Mon Oct 12 15:41:25 2009 From: allan.jones from gmx.de (Allan) Date: Mon Oct 12 15:52:20 2009 Subject: (kein Betreff) Message-ID: <4AFB19D0.1090209@gmx.de> Hi! I'm back to the old ethidium topic again! I have been working with this chemical a lot over the past two years and have been wondering how mutagenic/carcinogenic/teratogenic it actually is in the body. I can't imagine whether it will be resorbed via the skin into the blood system or whether a polar molecule of this size will cross the blood-brain barrier/seratoli barrier/placenta. Don't get me wrong, I am careful with this substance, but being quite a worryful person, I am sometimes worried that somewhere in the lab there could be contamination from coworkers (you can't always run around with a UV light) Different people in the lab treat the chemical with different degrees of caution and I just wonder whether microgram quantities present a serious risk or if they are rather more comparable to smoking a cigarette. I sometimes ask myself whether the amount of hysteria made around etbr is justified when chemicals like formaldehyde, phenol and chloroform are also used in the lab. Does anyone have a clue as to the risk of this chemical (in comparision to say a cigarette), especially in terms of crossing the placenta etc. and do you know how long it is stable in normal light? Thanks for any answers, as I am getting a little worried about the substance! All the best, Allan From rory.obrien from otago.ac.nz Mon Oct 12 15:32:48 2009 From: rory.obrien from otago.ac.nz (Rory O'Brien) Date: Mon Oct 12 15:52:30 2009 Subject: 3' end labelling with tritiated thymidine... In-Reply-To: References: Message-ID: <018b01ca4b7b$2984f380$7c8eda80$@obrien@otago.ac.nz> Cheers Duncan, The question came from a student here who wants to measure uptake of an oligo into bacterial cells and the effect of a drug on same. The thought was to label the oligo and measure via scintillation counter. I had thought we could end tail the oligo with tritiated thymidine but she's now wary of sticking a whole bunch of nucleotides onto the end of the oligo and the effect that might have on her uptake experiment. I doubt we'd get a strong enough signal with just end labeling and I'd rather avoid P32 as we're not really set up for it here. Could we do something non-isotopic with digoxygenin do you think? Maybe a dot blot of lysed bugs and semi-quantitative chemiluminescent detection? - Rory Historians believe that in newspost on Fri, 9 Oct 2009, Rory O'Brien penned the following literary masterpiece: >Could anyone maybe suggest which thymidine product might be most appropriate >to this application? Detection to be via scintillation counter; one for the >old-school molecular biologists maybe ;-) Highest specific activity would be best so either of the last two. Whether they are the least expensive is another matter. If you are simply using it to check the incorporation even the first one should do. You will just see lower counts. Duncan -- I love deadlines. I especially like the whooshing noise they make as they go flying by. Duncan Clark GeneSys Ltd. _______________________________________________ Methods mailing list Methods@net.bio.net http://www.bio.net/biomail/listinfo/methods From R.Jayakumar from roswellpark.org Mon Oct 12 16:25:17 2009 From: R.Jayakumar from roswellpark.org (Jayakumar, R) Date: Mon Oct 12 16:28:07 2009 Subject: (kein Betreff) In-Reply-To: <4AFB19D0.1090209@gmx.de> References: <4AFB19D0.1090209@gmx.de> Message-ID: Well.. I once spilled a 10mg/ml ethidium bromide a couple of ml on my palm and I am perfectly healthy and have had a normal life so far. Me and my wife have been working with ethidium bromide for more than a decade now and we have perfectly normal and healthy children. Just be careful but don't get hysteric if it falls on your hand. Follow the recommendations for cleaning it off as soon as possible. It is not that dangerous as people say it is, but I would take every precaution, lest I become the first guinea pig. I guess from my experience, a spill once or twice inyour life time should be fine :-))) It is mostly hysteria. Anyway, there are no studies showing it to be a carcinogen in humans yet. So it is always a "suspected" carcinogen / mutagen due to its DNA intercalating properties. It defenitely is safer than cigarrette smoke (proven carcinogen). Bye and best of luck in handling. Jay -----Original Message----- From: methods-bounces@oat.bio.indiana.edu [mailto:methods-bounces@oat.bio.indiana.edu] On Behalf Of Allan Sent: Wednesday, November 11, 2009 3:09 PM To: methods@magpie.bio.indiana.edu Subject: (kein Betreff) Hi! I'm back to the old ethidium topic again! I have been working with this chemical a lot over the past two years and have been wondering how mutagenic/carcinogenic/teratogenic it actually is in the body. I can't imagine whether it will be resorbed via the skin into the blood system or whether a polar molecule of this size will cross the blood-brain barrier/seratoli barrier/placenta. Don't get me wrong, I am careful with this substance, but being quite a worryful person, I am sometimes worried that somewhere in the lab there could be contamination from coworkers (you can't always run around with a UV light) Different people in the lab treat the chemical with different degrees of caution and I just wonder whether microgram quantities present a serious risk or if they are rather more comparable to smoking a cigarette. I sometimes ask myself whether the amount of hysteria made around etbr is justified when chemicals like formaldehyde, phenol and chloroform are also used in the lab. Does anyone have a clue as to the risk of this chemical (in comparision to say a cigarette), especially in terms of crossing the placenta etc. and do you know how long it is stable in normal light? Thanks for any answers, as I am getting a little worried about the substance! All the best, Allan _______________________________________________ Methods mailing list Methods@net.bio.net http://www.bio.net/biomail/listinfo/methods This email message may contain legally privileged and/or confidential information. If you are not the intended recipient(s), or the employee or agent responsible for the delivery of this message to the intended recipient(s), you are hereby notified that any disclosure, copying, distribution, or use of this email message is prohibited. If you have received this message in error, please notify the sender immediately by e-mail and delete this email message from your computer. Thank you. From cathalgarvey from gmail.com Tue Oct 13 06:17:21 2009 From: cathalgarvey from gmail.com (Cathal Garvey) Date: Tue Oct 13 08:39:00 2009 Subject: (kein Betreff) In-Reply-To: References: <4AFB19D0.1090209@gmx.de> Message-ID: <468b1a400910130417n75c440dboc352954240990be0@mail.gmail.com> I'd agree: Although it's not as dangerous as it's often made out to be, that doesn't mean you shouldn't be as careful as you're being. Put it like this; if you want to think of it like a cigarette, that might make a lot of sense. A normal person exposed once or twice to miniscule quantities probably won't notice the effects over the background noise of life's health problems. However a researcher should be careful to limit exposure because they'll be exposed constantly, like a life-long smoker if you like. They're more likely to suffer long term effects because, although it's not *that* toxic, the additive impact of being dosed so many times over your lifetime do add up. Not to mention; it's always the same part that gets dosed; the hands and face (from unwashed hands) in cases of poor handling. That's a risk factor in itself. Also, it is apparently readily absorbed through skin, despite its polarity. So I would assume to be on the safe side that it can cross the placenta or blood/brain, too, to be safe. From blackhole from abuse.plus.com Tue Oct 13 08:14:18 2009 From: blackhole from abuse.plus.com (Duncan Clark) Date: Tue Oct 13 12:39:11 2009 Subject: PhiX174 resistance E. coli strain? References: <200910121703.n9CH3wM16797@net.bio.net> Message-ID: Historians believe that in newspost on Mon, 12 Oct 2009, Yoram Gerchman penned the following literary masterpiece: >So EcoB and EcoK are >Type I restriction enzymes and will degrade the phiX174 genome Only if the genome contains a recognition site for those enzymes. If it doesn't I can't see offhand (bar any particular gene requirement) why it shouldn't infect a B or K12 strain. I wonder if the very first papers on phiX174 show strain information with regards E.coli C, K12 and B. Quite often that was one thing that was published, especially for say T-odd or T-even phages. Bit more digging required I think :-) Duncan -- I love deadlines. I especially like the whooshing noise they make as they go flying by. Duncan Clark GeneSys Ltd. From blackhole from abuse.plus.com Tue Oct 13 08:16:27 2009 From: blackhole from abuse.plus.com (Duncan Clark) Date: Tue Oct 13 12:39:18 2009 Subject: 3' end labelling with tritiated thymidine... References: Message-ID: <3m4ggYfr2H1KFA$p@abuse.plus.com> Historians believe that in newspost on Tue, 13 Oct 2009, Rory O'Brien penned the following literary masterpiece: >Could we do something non-isotopic with digoxygenin do you think? Problem then is, will an oligo with such a big group stuck on the end go into the cell and if so will such a big group alter the experimental outcome. Why not kinase an oligo with gamma labelled 32P ATP? Duncan -- I love deadlines. I especially like the whooshing noise they make as they go flying by. Duncan Clark GeneSys Ltd. From taliaferrod from mail.nih.gov Tue Oct 13 09:59:27 2009 From: taliaferrod from mail.nih.gov (Taliaferro, Dwayne (NIH/NIMH) [F]) Date: Tue Oct 13 12:39:25 2009 Subject: (kein Betreff) In-Reply-To: <4AFB19D0.1090209@gmx.de> Message-ID: I read somewhere that EtBr can cross through latex gloves (nytrile gloves are safe)---just to add to the hysteria. :P On 11/11/09 4:08 PM, "Allan" wrote: Hi! I'm back to the old ethidium topic again! I have been working with this chemical a lot over the past two years and have been wondering how mutagenic/carcinogenic/teratogenic it actually is in the body. I can't imagine whether it will be resorbed via the skin into the blood system or whether a polar molecule of this size will cross the blood-brain barrier/seratoli barrier/placenta. Don't get me wrong, I am careful with this substance, but being quite a worryful person, I am sometimes worried that somewhere in the lab there could be contamination from coworkers (you can't always run around with a UV light) Different people in the lab treat the chemical with different degrees of caution and I just wonder whether microgram quantities present a serious risk or if they are rather more comparable to smoking a cigarette. I sometimes ask myself whether the amount of hysteria made around etbr is justified when chemicals like formaldehyde, phenol and chloroform are also used in the lab. Does anyone have a clue as to the risk of this chemical (in comparision to say a cigarette), especially in terms of crossing the placenta etc. and do you know how long it is stable in normal light? Thanks for any answers, as I am getting a little worried about the substance! All the best, Allan _______________________________________________ Methods mailing list Methods@net.bio.net http://www.bio.net/biomail/listinfo/methods From cathalgarvey from gmail.com Wed Oct 14 03:59:54 2009 From: cathalgarvey from gmail.com (Cathal Garvey) Date: Wed Oct 14 10:18:37 2009 Subject: (kein Betreff) In-Reply-To: References: <4AFB19D0.1090209@gmx.de> Message-ID: <468b1a400910140159l1172fff9icae8f906480b340a@mail.gmail.com> Indeed, so they say in our lab too! I don't worry too much about that though unless there's a spillage, or until I start handling the gel itself. I just don't see there being *enough* Bromide on any dry, normal surfaces to meaningfully impact glove safety. For Gel Work, I do use the Nitrile gloves though. Interestingly, I just read through the Sybr-Safe manual; they had an independent lab compare the two for safety, and had a fold-difference chart for mutagenicity. In most cases, EtBr didn't do much. For certain strains tested though (not sure what the "Strains" were, but they were mammalian I believe) the fold difference was over 40 for EtBr. So it can be a significant mutagen in some circumstances all right. From cathalgarvey from gmail.com Thu Oct 15 10:32:21 2009 From: cathalgarvey from gmail.com (Cathal Garvey) Date: Thu Oct 15 13:42:24 2009 Subject: Peptide/Amino Fusion by Chemistry Message-ID: <468b1a400910150832o71d5a289t56e041ed76a88a06@mail.gmail.com> Hello all, I'm trying to puzzle out the practicality of a standardised method for peptide fusion in vitro. Basically, to take two peptides or amino backbones, and fuse one to the other chemically somehow. Assuming both could be arranged to have uncomplicated N/C terminal ends, how does one go about bonding them together? Is it reliable? Easy? Many thanks, Cathal Garvey From engelbert_buxbaum from hotmail.com Thu Oct 15 13:50:37 2009 From: engelbert_buxbaum from hotmail.com (Dr Engelbert Buxbaum) Date: Thu Oct 15 14:59:24 2009 Subject: (kein Betreff) References: Message-ID: Am 13.10.2009, 10:59 Uhr, schrieb Taliaferro, Dwayne (NIH/NIMH) [F] : > I sometimes ask myself whether the amount of hysteria made around etbr > is justified when chemicals like formaldehyde, phenol and chloroform are > also used in the lab. Now, that would require knowledge about the dangers of those chemicals. Phenol was used for decades as disinfectant for surgery, a fine mist of the aqueous solution was sprayed onto patients, physicians and nurses alike. To the best of my knowledge at least this long-term exposure of medical personal did not lead to an increase in cancer or in flk (funny looking kids). Chloroform was also used during surgery, as a volatile anaestetic. Although its flammability has indeed caused some problems (not quite as bad as those with ether, though), I am not aware of any epidemiological studies showing problems in medical personnel. And formaldehyde has been "shown" to be carcinogenic in rats by exposing the poor critters to concentrations of the stuff that no human could possibly tolerate. But the rats were in a cage and couldn't run away. The resulting cross-linked proteins in their respiratory tract (remember, even though formaldehyde has only one aldehyde group, it is a bifunctional cross-linker) led to chronic immune activation, and /that/ is a well-known cause of cancer. Again, epidemiology amongst pathologists (who used to use the stuff by the drum) has never shown increased morbidity or mortality. That does not mean that formaldehyde can not act as an allergen, though. Work cleanly, avoid exposure as much as reasonably possible, don't break the law. But be aware that many experimental scientist before you have lived long and healthy lifes. From nick.theodorakis from gmail.com Thu Oct 15 13:55:42 2009 From: nick.theodorakis from gmail.com (Nick Theodorakis) Date: Thu Oct 15 14:59:30 2009 Subject: Peptide/Amino Fusion by Chemistry References: Message-ID: <349969c9-87ee-4925-8b65-917afedeb929@j4g2000yqa.googlegroups.com> On Oct 15, 10:32?am, Cathal Garvey wrote: > Hello all, > I'm trying to puzzle out the practicality of a standardised method for > peptide fusion in vitro. Basically, to take two peptides or amino backbones, > and fuse one to the other chemically somehow. Assuming both could be > arranged to have uncomplicated N/C terminal ends, how does one go about > bonding them together? Is it reliable? Easy? > > Many thanks, > Cathal Garvey There are several ways that are used to make synthetic peptides; you can review then at the wikipedia article here: Is this similar to what you had in mind, or were you thinking about something different? Nick -- Nick Theodorakis nick_theodorakis@hotmail.com contact form: http://theodorakis.net/contact.html From novalidaddress from nurfuerspam.de Thu Oct 15 14:30:42 2009 From: novalidaddress from nurfuerspam.de (WS) Date: Thu Oct 15 14:59:35 2009 Subject: Peptide/Amino Fusion by Chemistry References: Message-ID: Hi Cathal, if there are no reactive side chains and you "just" want to join the carboxy terminus to the amino terminus (i.e. generate a cyclic peptide), then it might be easy with EDC and NHS in aqueous / buffered solution. Don't use amine and carboxylate based buffers of course (e.g. tris, acetate etc), as they will quench your reagents. Phosphate or borate should work. After the reaction, cleanup may be done with gel filtration, dialysis or an organic / aqueous extraction, depending on the size of the peptide / protein. Detailed adaptable protocols should be available from PubMed and / or Google by searching for "EDC" and "NHS". People use this approach usually for linking haptens to carrier proteins like BSA, KLH or to label antibodies and proteins with dye molecules. However, if you plan to fuse two peptides (both with an N and a C terminus) and / or have reactive side groups (lysine, glutamate, aspartate), then best ask a dedicated lab for assistance. You'll need a lot of protection (and subsequently deprotection) chemistry. Good luck! HTH Wo From cathalgarvey from gmail.com Fri Oct 16 03:14:52 2009 From: cathalgarvey from gmail.com (Cathal Garvey) Date: Fri Oct 16 10:13:30 2009 Subject: Peptide/Amino Fusion by Chemistry In-Reply-To: References: Message-ID: <468b1a400910160114s3e07b2d8s6a8a035e62e7287e@mail.gmail.com> Thanks for the detailed replies! After doing some reading yesterday on the matter, it seems far less simple than I'd thought it would be. To refine what I was asking, what I'm looking at doing is using a synthesised strand of Peptide Nucleic Acid, which would have an N and C terminus and would probably lack protection (aside from whatever blocking groups were used to build the chain). To that I'd aim to fuse a protein produced in the lab by e.coli, which I think is expected to have natural protection or blocking groups on either terminus (?). I have read a little about inteins, which seem to be a clever way of having protective groups cut themselves off. If they work well enough to generate a large quantity of unprotected protein, it should mean both of my fragments (PNA and protein) are unprotected at the relevant termini. So, for unprotected termini and straightforward N-C fusion, what would you recommend as an in-lab fusion reaction? Please note that I'm a molecular biologist by trade, rather than an organic chemist. From davidminde from gmail.com Fri Oct 16 11:06:35 2009 From: davidminde from gmail.com (David-Paul Minde) Date: Fri Oct 16 14:33:15 2009 Subject: Peptide/Amino Fusion by Chemistry In-Reply-To: <468b1a400910160114s3e07b2d8s6a8a035e62e7287e@mail.gmail.com> References: <468b1a400910160114s3e07b2d8s6a8a035e62e7287e@mail.gmail.com> Message-ID: <123243900910160906v5393da77s26ac476b6061ffac@mail.gmail.com> Hi Cathal, I think the straightforward way is using sortase a reaction for your fusions: one c-terminus has LPXTGXX motif and is fused to another n-terminus via sortase a enzyme. The beauty of the system is specificity, short recognition motifs (no add. cloning required ;) virtually no influence on expression of target proteins, compatibility with diverse coupling chemistries inlcuding PNAs according to an exploding literature in recent years. Sortase a can be easily expressed in bacteria. Introduction of "LPXTGGG" linker motif may be the only major disadvantage. Intein is also fine in some cases and more puristic chemistry. However, extreme variability of efficiency generally "kills" many intein fusion attempts. cheers, David 2009/10/16 Cathal Garvey > Thanks for the detailed replies! > After doing some reading yesterday on the matter, it seems far less simple > than I'd thought it would be. > > To refine what I was asking, what I'm looking at doing is using a > synthesised strand of Peptide Nucleic Acid, which would have an N and C > terminus and would probably lack protection (aside from whatever blocking > groups were used to build the chain). To that I'd aim to fuse a protein > produced in the lab by e.coli, which I think is expected to have natural > protection or blocking groups on either terminus (?). > > I have read a little about inteins, which seem to be a clever way of having > protective groups cut themselves off. If they work well enough to generate > a > large quantity of unprotected protein, it should mean both of my fragments > (PNA and protein) are unprotected at the relevant termini. > > So, for unprotected termini and straightforward N-C fusion, what would you > recommend as an in-lab fusion reaction? Please note that I'm a molecular > biologist by trade, rather than an organic chemist. > _______________________________________________ > Methods mailing list > Methods@net.bio.net > http://www.bio.net/biomail/listinfo/methods > -- David Minde MSc (TUM) Cellular Protein Chemistry Room 707 Department of Chemistry Faculty of Science Utrecht University Krytgebouw Padualaan 8 NL-3584 CH Utrecht The Netherlands office phone +31 30 253 4105 (magic:) android +31 652614443 home: Griftkade 4 bis 3572 TW Utrecht Science is what happens while we are making other plans (~John Lennon) From m.ramirez from ucl.ac.uk Sat Oct 17 09:37:38 2009 From: m.ramirez from ucl.ac.uk (Manfred Junemann Ramirez) Date: Sat Oct 17 16:11:37 2009 Subject: Lentiviral Vector Message-ID: Hi, has abyone here some experience with lentiviral vectors for bone marrow stem cell transduction? I am trying to work out optimum MOI and general transduction methods: spin occulation etc... regards manfred From stewjw from gmail.com Mon Oct 19 03:14:01 2009 From: stewjw from gmail.com (StewJW) Date: Mon Oct 19 10:35:00 2009 Subject: Peptide/Amino Fusion by Chemistry References: <468b1a400910160114s3e07b2d8s6a8a035e62e7287e@mail.gmail.com> Message-ID: A good start would be to drop the fusion terminology and define where you want your covalent linkage formed. If your going to go down the synthetic route I'd seek advice from a peptide chemist. If you just want to perfom a simple cross-linking reaction Pierce has one the most comprehensive range of reagents around. They also have a handbook which offers advice: http://www.piercenet.com/Objects/View.cfm?Type=Page&ID=FE7F690D-58AE-4342-AE85-BA94DCA642F8 From spsen from ou.ac.lk Mon Oct 19 03:00:49 2009 From: spsen from ou.ac.lk (Prasad Senadheera) Date: Mon Oct 19 10:35:26 2009 Subject: insitue PCR Message-ID: <20091019075853.M50985@samanala.ou.ac.lk> Hi all I am rlooking forward to gettng done insitue pcr for a plant tissue - rice root- to detect the expression of a gene. since there are no facilities in here can any of you let me know of any person / organisation that carry on insitue pcr at a cost? cheers Prasad -- This message has been scanned for viruses and dangerous content by OUSL E-Mail Scanner, and is believed to be clean. From pradeepiyer from heterodrugs.com Mon Oct 19 23:26:25 2009 From: pradeepiyer from heterodrugs.com (Mr.Pradeep Kumar Iyer Group Leader, Bio Tech, HRF) Date: Tue Oct 20 14:00:43 2009 Subject: Querry regarding silver staining for glycoproteins Message-ID: <612b9b830910192126y4543a7bei502a574db6c2601d@mail.gmail.com> Dear All, I have this confusion on the silver staining of glycoproteins in IEF gels. I have observed that with increasing glycosylation, the intensity of the glycoproteins after silver stain decreases. I attribute this to the masking of the aminoacids by the glycan chain resulting in the decrease in efficiency of the silver stain. Has anyone done an IEF for deglycosylated molecule? Does it give a more intense band than the glycosylated one? My next experiment would be that but if that is observed by most of you, I would not waste my resources on doing that. Kindly give some inputs. Regards, Pradeep Iyer --=20 If you're not part of the solution, you're part of the precipitate =96 Henr= y J. Tillman From adrbaha from gmail.com Tue Oct 20 18:05:08 2009 From: adrbaha from gmail.com (Aqua Gem) Date: Tue Oct 20 22:53:34 2009 Subject: PCR Lab Design Message-ID: <000901ca51d9$f4b7f4d0$de27de70$@com> Dear Mr. Carson, Please allow me to introduce myself. My name is Andrea Budd and I am working for the Belize Agricultural Health Authority as the Coordinator of the Aquatic Health Management Unit. A major component of this management unit is to set up and have functional diagnostic labs for disease monitoring and by extension - farm certification. I have been searching the net for any information I could find on planning and designing a PCR lab and it was through this means I came across your message regarding the same topic (at www.bio.net/). Though it seems this message is a bit dated I am hoping to make contact with you and ask for some insight on PCR Lab design. Grateful if you could respond. Sincerely, Andrea Budd. -- Andrea Budd (Mrs.) Aquatic Health Unit Animal Health Department Belize Agricultural Health Authority Tel: (501) 224-4794/223-4457 Fax: (501) 224-5230 From RayHL from gmx.de Wed Oct 21 07:36:08 2009 From: RayHL from gmx.de (RayHL@gmx.de) Date: Wed Oct 21 08:33:08 2009 Subject: JM101 for Protein Expression? Message-ID: <20091021123608.199270@gmx.net> Hi everyone, I'm planning some protein expressions and right now, I'm choosing my expression strains. I'm wondering about the ability of JM101 cells to express recombinant proteins. At my old lab, we used them for some expressions (and it did work fine), but after checking the genotype, they don't seem to lack proteases like ompT or lon, and they don't seem to have T7 oR T5 RNA polymerase. So, the question is: why did they work at all? Or was it just a labeling error and we never really used JM101? Thanks for your answers in advance! Ray -- Jetzt kostenlos herunterladen: Internet Explorer 8 und Mozilla Firefox 3.5 - sicherer, schneller und einfacher! http://portal.gmx.net/de/go/chbrowser From davidminde from gmail.com Wed Oct 21 10:45:45 2009 From: davidminde from gmail.com (David-Paul Minde) Date: Wed Oct 21 11:54:25 2009 Subject: JM101 for Protein Expression? In-Reply-To: <20091021123608.199270@gmx.net> References: <20091021123608.199270@gmx.net> Message-ID: <123243900910210845l556e93ccqc88a3e6ac1e224d7@mail.gmail.com> not all proteins are proteolytically unstable (e.g. GFP is rather robust once it is folded) most proteins are easy to degrade, however, because there is usually some flexible part which is easily accessible for proteolysis ... D 2009/10/21 > Hi everyone, > > I'm planning some protein expressions and right now, I'm choosing my > expression strains. I'm wondering about the ability of JM101 cells to > express recombinant proteins. At my old lab, we used them for some > expressions (and it did work fine), but after checking the genotype, they > don't seem to lack proteases like ompT or lon, and they don't seem to have > T7 oR T5 RNA polymerase. > > So, the question is: why did they work at all? Or was it just a labeling > error and we never really used JM101? > > Thanks for your answers in advance! > > > Ray > -- > Jetzt kostenlos herunterladen: Internet Explorer 8 und Mozilla Firefox 3.5 > - > sicherer, schneller und einfacher! http://portal.gmx.net/de/go/chbrowser > > _______________________________________________ > Methods mailing list > Methods@net.bio.net > http://www.bio.net/biomail/listinfo/methods > -- David Minde MSc (TUM) Cellular Protein Chemistry Room 707 Department of Chemistry Faculty of Science Utrecht University Krytgebouw Padualaan 8 NL-3584 CH Utrecht The Netherlands office phone +31 30 253 4105 (magic:) android +31 652614443 Science is what happens while we are making other plans (~John Lennon) From R.Jayakumar from roswellpark.org Wed Oct 21 13:06:17 2009 From: R.Jayakumar from roswellpark.org (Jayakumar, R) Date: Wed Oct 21 15:32:16 2009 Subject: JM101 for Protein Expression? In-Reply-To: <20091021123608.199270@gmx.net> References: <20091021123608.199270@gmx.net> Message-ID: That shouldn't be a problem for expression in E.coli as long as it is recognized by native RNA polymerases. Remember the plasmid antibiotic genes used for selection is recognized and expressed, so will other e.coli promoters used to express your gene. For T7 to work, you will need an E.coli strain with the T7 phage genome as a lysogen expressing the viral T7 RNA polymerase, such as XL1blue-DE3. best of luck Jay -----Original Message----- From: methods-bounces@oat.bio.indiana.edu [mailto:methods-bounces@oat.bio.indiana.edu] On Behalf Of RayHL@gmx.de Sent: Wednesday, October 21, 2009 8:36 AM To: methods@magpie.bio.indiana.edu Subject: JM101 for Protein Expression? Hi everyone, I'm planning some protein expressions and right now, I'm choosing my expression strains. I'm wondering about the ability of JM101 cells to express recombinant proteins. At my old lab, we used them for some expressions (and it did work fine), but after checking the genotype, they don't seem to lack proteases like ompT or lon, and they don't seem to have T7 oR T5 RNA polymerase. So, the question is: why did they work at all? Or was it just a labeling error and we never really used JM101? Thanks for your answers in advance! Ray -- Jetzt kostenlos herunterladen: Internet Explorer 8 und Mozilla Firefox 3.5 - sicherer, schneller und einfacher! http://portal.gmx.net/de/go/chbrowser _______________________________________________ Methods mailing list Methods@net.bio.net http://www.bio.net/biomail/listinfo/methods This email message may contain legally privileged and/or confidential information. If you are not the intended recipient(s), or the employee or agent responsible for the delivery of this message to the intended recipient(s), you are hereby notified that any disclosure, copying, distribution, or use of this email message is prohibited. If you have received this message in error, please notify the sender immediately by e-mail and delete this email message from your computer. Thank you. From allan.jones from gmx.de Thu Oct 22 09:45:26 2009 From: allan.jones from gmx.de (Allan Jones) Date: Thu Oct 22 11:52:50 2009 Subject: chloroform Message-ID: <20091022144526.273680@gmx.net> Hi! I just accidently poured some concentrated chloroform over my (nitril) gloves. I took them off as quickly as possible and washed my hands, but how dangerous is this actually (terato-/carcinogen???) all the best, al -- GRATIS f?r alle GMX-Mitglieder: Die maxdome Movie-FLAT! Jetzt freischalten unter http://portal.gmx.net/de/go/maxdome01 From novalidaddress from nurfuerspam.de Thu Oct 22 12:52:47 2009 From: novalidaddress from nurfuerspam.de (WS) Date: Thu Oct 22 13:49:57 2009 Subject: chloroform References: Message-ID: <22d44247-f323-4aab-a6ac-8ed9aa89265c@g1g2000vbr.googlegroups.com> Hi Allen, don't worry. But if you think you feel unwell, see a doctor. Not so long ago, it even was used in medicine as narcotic. Organic chemists routinely use it as a convenient solvent and probably breath a lot of it and sometimes even pour it over their hands. Chronic exposure will harm your liver however. You might have a look here: http://en.wikipedia.org/wiki/Chloroform Have fun, Wo From ivanoov from gmail.com Fri Oct 23 02:20:02 2009 From: ivanoov from gmail.com (chovek69) Date: Fri Oct 23 10:44:26 2009 Subject: chloroform References: <22d44247-f323-4aab-a6ac-8ed9aa89265c@g1g2000vbr.googlegroups.com> Message-ID: On Oct 22, 8:52?pm, WS wrote: > Hi Allen, > > don't worry. But if you think you feel unwell, see a doctor. > > Not so long ago, it even was used in medicine as narcotic. Organic > chemists routinely use it as a convenient solvent and probably breath > a lot of it and sometimes even pour it over their hands. Chronic > exposure will harm your liver however. > > You might have a look here:http://en.wikipedia.org/wiki/Chloroform > > Have fun, > > Wo Don't bother at all. You are safe. From engelbert_buxbaum from hotmail.com Fri Oct 23 10:29:55 2009 From: engelbert_buxbaum from hotmail.com (Dr Engelbert Buxbaum) Date: Fri Oct 23 11:13:46 2009 Subject: Peptide/Amino Fusion by Chemistry References: Message-ID: Am 16.10.2009, 04:14 Uhr, schrieb Cathal Garvey : > To refine what I was asking, what I'm looking at doing is using a > synthesised strand of Peptide Nucleic Acid, which would have an N and C > terminus and would probably lack protection (aside from whatever blocking > groups were used to build the chain). To that I'd aim to fuse a protein > produced in the lab by e.coli, which I think is expected to have natural > protection or blocking groups on either terminus (?). It would probably be a lot easier to make a new DNA construct and express that. The chemistry involved in your fusion reaction might be feasible, if complicated, in peptides. However, it would almost certainly destroy the secondary and tertiary structure of a protein. If you could live with linking peptide (e.g. a hapten) and protein (as carrier) at some reactive residue, rather then a C-to-N linkage, that would be a lot simpler, and there are a lot of tried procedures out there (think HRP-antibody conjugates). From hroychow from nmsu.edu Sat Oct 24 20:17:55 2009 From: hroychow from nmsu.edu (Dr. Hiranya S. Roychowdhury) Date: Sat Oct 24 21:37:11 2009 Subject: chloroform In-Reply-To: <20091022144526.273680@gmx.net> References: <20091022144526.273680@gmx.net> Message-ID: <2224.67.41.10.197.1256433475.squirrel@webmail.nmsu.edu> Relax! Not that bad! I have had many a spill of chloroform on my hands, and nitril gloves do not dissolve as quickly in that solvent. > Hi! > > I just accidently poured some concentrated chloroform over my (nitril) > gloves. I took them off as quickly as possible and washed my hands, but > how dangerous is this actually (terato-/carcinogen???) > > all the best, > al > -- > GRATIS f?r alle GMX-Mitglieder: Die maxdome Movie-FLAT! > Jetzt freischalten unter http://portal.gmx.net/de/go/maxdome01 > > _______________________________________________ > Methods mailing list > Methods@net.bio.net > http://www.bio.net/biomail/listinfo/methods > -- Hiranya S. Roychowdhury, Ph.D. Asst. Professor, Health & Public Services Dona Ana Community College New Mexico State University Las Cruces, NM 88003 From rahimpour_a from yahoo.com Sun Oct 25 09:16:01 2009 From: rahimpour_a from yahoo.com (Azam Rahimpour) Date: Sun Oct 25 11:07:51 2009 Subject: anti Tag antibody cross reactivity Message-ID: <828417.76415.qm@web52705.mail.re2.yahoo.com> Hi does anybody use different epitope tags for proteins expressed in mammalian cells? I have some questions about this: ? 1. Is it important to add small tags to either C or N terminal of proteins? ? 2. Is it necessary to put an spacer or specific sequence between tag and protein coding region? ? 3. which anti tag (FLAG, His, myc, HA ...) has lower cross reactivity to cellular proteins? ? thanks in advance From editor from gene-quantification.info Sun Oct 25 10:36:51 2009 From: editor from gene-quantification.info (Editor www.Gene-Quantification.info) Date: Sun Oct 25 11:07:58 2009 Subject: International qPCR 2010 Symposium & Exhibition in Vienna Message-ID: <689beb7c-c640-46b6-ad40-e04c17e0fb0e@m1g2000vbi.googlegroups.com> International qPCR 2010 Symposium & Exhibition in Vienna 7th =96 9th April 2010 The focus of the qPCR 2010 Event will be =93The ongoing evolution of qPCR=94 http://www.qpcr2010-vienna.net/ qPCR2010@BioEPS.com --------------------------------------------------------------------------- qPCR 2010 in Vienna International qPCR 2010 Symposium and Exhibition in Vienna 7th =96 9th April 2010 The focus of the qPCR 2010 Event will be =93The ongoing evolution of qPCR=94 representing all new and emerging techniques, applications and data analysis methods. MIQE, HRM, microRNA, CNV, single-cell qPCR, digital PCR, and analysis of circulating nucleic acids will be in the focus of the conference =3D> http://www.qpcr2010-vienna.net/ qPCR Symposium topic - =93The ongoing evolution of qPCR=94 The symposium focus will be on 40 lectures presented by international recognized experts in their application fields. The emphasis will be on unbiased, didactic information exchange. One third of the talks will be presented by invited speakers, one third of the speakers will be selected from the submitted abstracts and one third will be qPCR company representatives with their newest qPCR technologies. The focus of the qPCR 2010 Event will be =93The ongoing evolution of qPCR=94 representing all new and emerging techniques, applications and data analysis methods: Symposium sessions & preliminary titles MIQE and QM strategies in qPCR Prof. Stephen Bustin, =93The MIQE Guidelines: Minimum Information for Publication of Quantitative Real-Time PCR Experiments=94 High throughput quantitative PCR =96 digital PCR Prof. Mikael Kubista, =93Digital PCR and intracellular expression profiling=94 Dr. Philip Day, =93High throughput droplet PCR=94 Dr. Ken Livak HRM =96 High Resolution Melting - Epigenetics Prof. Carl Wittwer, "High Resolution Melting Analysis" Prof. Claudio Orlando, =93High Resolution Melting Analysis in Cancer Diagnosis=94 Circulating nucleic acids Dr. Pamela Pinzani, =93Cell free circulating DNA=94 Dr. Alfred Sch=F6ller, =84Targeting the human urine RNAome for tumor diagnostics by qPCR=94 Single-cell qPCR Dr. Michael W. Pfaffl, =93Quantitative expression analysis after pre-amp in single WBCs=94 Dr. Anders Stahlberg, =93Single-cell gene expression RNAi - microRNA - siRNA Applications =96 miRNA normalisation Prof. Jo Vandesompele, =93MicroRNA and mRNA gene expression normalization=94 Dr. Mirco Castoldi, "Expression profiling of microRNA by quantitative real time PCR, what is available and where to go from there" qPCR BioStatistics & BioInformatics Dr. Ales Tichopad, =93Statistical aspects of quantitative PCR experiment design and qPCR data analysis=94 Dr. Jan Hellemans, =93Accurate and objective copy number profiling using real-time quantitative PCR=94 Dr. Anders Bergkvist, =93Expression profiling - clusters of possibilities=94 -------------------------------------------------------- For more information: Michael Pfaffl BioEPS GmbH Lise Meitner Strasse 30 85354 Freising Weihenstephan Germany qPCR2010@BioEPS.com From Vince.Mulholland from sasa.gsi.gov.uk Mon Oct 26 04:07:32 2009 From: Vince.Mulholland from sasa.gsi.gov.uk (Vince Mulholland) Date: Mon Oct 26 11:53:50 2009 Subject: PCR Lab Design Message-ID: Dear Andrea, In case you didn't get a reply I have attached some links to relevant publications on PCR lab design (see below). I designed a PCR lab a few years ago, and the main constraints on your design will be the space available plus any budgetary limits. The important thing in the design of PCR work spaces is compartmentalization; at least 2 rooms are required, but if you are going to be doing large-scale routine PCR you'd probably need to set the lower limit at 3 rooms. Regards, Vince Links: www.hpa-standardmethods.org.uk/documents/qsop/pdf/qsop38.pdf www.biosupplynet.com/pdf/01_PCR_Primer_p.5_14.pdf genome.cshlp.org/content/3/2/S2.full.pdf Vincent Mulholland Molecular Biology Unit Manager - Diagnostics & Molecular Biology Section Science and Advice for Scottish Agriculture (SASA) SASA is a Division of the Scottish Government Rural Payments and Inspections Directorate Correspondents should note that all communications to or from SASA (Science and Advice for Scottish Agriculture ? a Division of the Rural Payments and Inspections Directorate of the Scottish Government) may be automatically logged, monitored and/or recorded for lawful purposes. The original of this email was scanned for viruses by the Government Secure Intranet (GSi) virus scanning service. On leaving the GSi this email was certified virus-free. From prae from gmx.net Mon Oct 26 05:25:00 2009 From: prae from gmx.net (Christian Praetorius) Date: Mon Oct 26 11:53:58 2009 Subject: Blue high MW electrophoresis tracking dye? References: Message-ID: <7klbnrF390b3mU1@mid.individual.net> Raymond Dalgleish wrote: >Any ideas about what the blue dye might have been in this incorrect >loading dye? This sounds very much like Xylene cyanol. Our loading biuffer contains both dyes. Christian -- X-no-Sig: yes From rwmd1 from NOSPAMle.ac.uk Mon Oct 26 04:19:13 2009 From: rwmd1 from NOSPAMle.ac.uk (Raymond Dalgleish) Date: Mon Oct 26 11:54:04 2009 Subject: Blue high MW electrophoresis tracking dye? Message-ID: Last week, a laboratory practical that I teach was ruined because our Central Teaching Unit provided blue loading dye that clearly did not migrate on 1% agarose with the 300 bp mobility expected of bromophenol blue. The dye was perhaps a little darker than BPB and appeared to have a mobility around 10 kb. The consequence was that all of the PCR products ran off the bottom of the gel. Any ideas about what the blue dye might have been in this incorrect loading dye? Many thanks, Raymond Dalgleish From bmacgreg from unc.edu Mon Oct 26 12:12:37 2009 From: bmacgreg from unc.edu (Barbara MacGregor) Date: Mon Oct 26 12:28:25 2009 Subject: Loading dye In-Reply-To: <200910261704.n9QH46M03272@net.bio.net> References: <200910261704.n9QH46M03272@net.bio.net> Message-ID: <5A206678-6348-4166-AA46-D59B88032BEA@unc.edu> Xylene cyanol definitely has a different color, though, at least as I've used it - blue-green rather than blue. But those are the only blue dyes I can think of, so that's probably it... Barbara > > Raymond Dalgleish wrote: > >> Any ideas about what the blue dye might have been in this incorrect >> loading dye? > > This sounds very much like Xylene cyanol. Our loading biuffer contains > both dyes. > > Christian > From R.Jayakumar from roswellpark.org Mon Oct 26 13:05:08 2009 From: R.Jayakumar from roswellpark.org (Jayakumar, R) Date: Mon Oct 26 15:46:41 2009 Subject: Loading dye In-Reply-To: <5A206678-6348-4166-AA46-D59B88032BEA@unc.edu> References: <200910261704.n9QH46M03272@net.bio.net> <5A206678-6348-4166-AA46-D59B88032BEA@unc.edu> Message-ID: Bromo phenol blue.. Frequently added with xylene cyanol in DNA loading dyes. Jay -----Original Message----- From: methods-bounces@oat.bio.indiana.edu [mailto:methods-bounces@oat.bio.indiana.edu] On Behalf Of Barbara MacGregor Sent: Monday, October 26, 2009 1:13 PM To: methods@oat.bio.indiana.edu Subject: Re: Loading dye Xylene cyanol definitely has a different color, though, at least as I've used it - blue-green rather than blue. But those are the only blue dyes I can think of, so that's probably it... Barbara > > Raymond Dalgleish wrote: > >> Any ideas about what the blue dye might have been in this incorrect >> loading dye? > > This sounds very much like Xylene cyanol. Our loading biuffer contains > both dyes. > > Christian > _______________________________________________ Methods mailing list Methods@net.bio.net http://www.bio.net/biomail/listinfo/methods This email message may contain legally privileged and/or confidential information. If you are not the intended recipient(s), or the employee or agent responsible for the delivery of this message to the intended recipient(s), you are hereby notified that any disclosure, copying, distribution, or use of this email message is prohibited. If you have received this message in error, please notify the sender immediately by e-mail and delete this email message from your computer. Thank you. From bzahedi from bccrc.ca Mon Oct 26 20:12:56 2009 From: bzahedi from bccrc.ca (Bari Zahedi) Date: Mon Oct 26 20:40:05 2009 Subject: MEG vs. PEG Message-ID: Has anyone used PEG to reduce melting temperature of their primers for PCR? We are working with very GC rich DNA and have been using Betane but we have read that MEG works better. We don't have any MEG but we do have PEG and were wondering if it could be substituted for MEG. Thanks in advance. From davidminde from gmail.com Mon Oct 26 21:06:44 2009 From: davidminde from gmail.com (David-Paul Minde) Date: Mon Oct 26 22:21:33 2009 Subject: MEG vs. PEG In-Reply-To: References: Message-ID: <123243900910261906u4f80136fy70c31be3c0201b8a@mail.gmail.com> how about DMSO ? if available, I think the additive of choice (because about everybody uses it and therefore tons of experience reports on it around). cheers, David 2009/10/27 Bari Zahedi > Has anyone used PEG to reduce melting temperature of their primers for PCR? > We are working with very GC rich DNA and have been using Betane but we have > read that MEG works better. We don't have any MEG but we do have PEG and > were wondering if it could be substituted for MEG. Thanks in advance. > > _______________________________________________ > Methods mailing list > Methods@net.bio.net > http://www.bio.net/biomail/listinfo/methods > -- David Minde MSc (TUM) Cellular Protein Chemistry Room 707 Department of Chemistry Faculty of Science Utrecht University Krytgebouw Padualaan 8 NL-3584 CH Utrecht The Netherlands office phone +31 30 253 4105 (magic:) android +31 652614443 home: Griftkade 4 bis 3572 TW Utrecht Science is what happens while we are making other plans (~John Lennon) From jason.d.fernandes from gmail.com Mon Oct 26 23:17:02 2009 From: jason.d.fernandes from gmail.com (Jason) Date: Tue Oct 27 07:57:38 2009 Subject: anti Tag antibody cross reactivity References: Message-ID: <3d540085-80ac-4a3c-85a1-a7873d496361@2g2000prl.googlegroups.com> Hi, Adding a small tag makes it easy to purify, IF, or id by western. In general a small flexible linker is a good idea GGGS is a standard linker but it will depend on how you clone it. N or C terminus will depend on your protein but in general there is not much of a difference (global TAP studies show little difference between N- terminal tagging and C-terminal, but if you know an important interaction occurs at one end you may want to avoid tagging it there). FLAG is considered a very clean tag, it is also very expensive reagent wise and depending on what you do you might denature the antibody (i.e no denaturing purification). His is a workhorse tag that can take a lot of abuse but you will purify polyhistidine proteins from mamallian extract. It is also very cheap. Strep II from IBA is a nice small clean tag that I use frequently. HA and myc are also fairly well established and generally considered cleaner than his but not as clean as FLAG (also in general I use 3xFLAG). Jason On Oct 25, 7:16?am, Azam Rahimpour wrote: > Hi > does anybody use different epitope tags for proteins expressed in mammalian cells? I have some questions about this: > ? > 1. Is it important to add small tags to either C or N terminal of proteins? > ? > 2. Is it necessary to put an spacer or specific sequence between tag and protein coding region? > ? > 3. which anti tag (FLAG, His, myc, HA ...) has lower cross reactivity to cellular proteins? > ? > thanks in advance From prae from gmx.net Tue Oct 27 04:30:20 2009 From: prae from gmx.net (Christian Praetorius) Date: Tue Oct 27 07:57:44 2009 Subject: Loading dye References: <200910261704.n9QH46M03272@net.bio.net> Message-ID: <7knstaF3akt33U1@mid.individual.net> Barbara MacGregor wrote: >Xylene cyanol definitely has a different color, though, at least as >I've used it - blue-green rather than blue. Thats right. But it migrates around 10k in a 1% gel. Christian -- X-no-Sig: yes From rahimpour_a from yahoo.com Tue Oct 27 09:28:41 2009 From: rahimpour_a from yahoo.com (Azam Rahimpour) Date: Tue Oct 27 11:01:56 2009 Subject: adding kozak sequence to primer Message-ID: <615744.83799.qm@web52705.mail.re2.yahoo.com> Hi I want to add kozak sequence to my primer for mamamlian expression but I know the consensus kozak sequence is gccgcc(a/g)ccAUGg but the second codon in my gene is started with a not g. should I convert it to g??to make it identical to kozak or using just gccgcc(a/g)ccAUG is OK? best From arnec from bio.umass.edu Tue Oct 27 12:09:23 2009 From: arnec from bio.umass.edu (Arne Christensen) Date: Tue Oct 27 16:43:48 2009 Subject: Custom ISH probe synthesis Message-ID: <4AE72943.4030502@bio.umass.edu> Dear Listeners, I am in a lab that does a great deal of protein work, but we have limited experience conducting molecular methods such as subcloning and ISH. We would like to run some ISH, and we are prepared to purchase the reagents to do so, however we are not in a position to set ourselves up for the molecular work underlying the generation of a plasmid for making RNA probes. So, we would like to purchase four RNA in situ probes. Two target probes with their respective sense negative controls. The sequences are very short, a 174-mer and a 71-mer. Can anybody recommend a commercial source for such RNA probe synthesis? Thank you in advance, Arne From R.Jayakumar from roswellpark.org Tue Oct 27 16:16:56 2009 From: R.Jayakumar from roswellpark.org (Jayakumar, R) Date: Tue Oct 27 16:43:57 2009 Subject: Loading dye In-Reply-To: <7knstaF3akt33U1@mid.individual.net> References: <200910261704.n9QH46M03272@net.bio.net> <7knstaF3akt33U1@mid.individual.net> Message-ID: Hmm.. XC I think moves with the 4K band and above the blue dye front of BPB (the blue dye he is talking about) moves with a 300 bp band on a 1% gel Jay -----Original Message----- From: methods-bounces@oat.bio.indiana.edu [mailto:methods-bounces@oat.bio.indiana.edu] On Behalf Of Christian Praetorius Sent: Tuesday, October 27, 2009 5:30 AM To: methods@magpie.bio.indiana.edu Subject: Re: Loading dye Barbara MacGregor wrote: >Xylene cyanol definitely has a different color, though, at least as >I've used it - blue-green rather than blue. Thats right. But it migrates around 10k in a 1% gel. Christian -- X-no-Sig: yes _______________________________________________ Methods mailing list Methods@net.bio.net http://www.bio.net/biomail/listinfo/methods This email message may contain legally privileged and/or confidential information. If you are not the intended recipient(s), or the employee or agent responsible for the delivery of this message to the intended recipient(s), you are hereby notified that any disclosure, copying, distribution, or use of this email message is prohibited. If you have received this message in error, please notify the sender immediately by e-mail and delete this email message from your computer. Thank you. From anibalara from gmail.com Tue Oct 27 21:06:07 2009 From: anibalara from gmail.com (=?utf-8?Q?An=C3=ADbal_Lara?=) Date: Wed Oct 28 10:36:25 2009 Subject: Cracking Preps for identifying inserts Message-ID: <04618D17-107E-4546-9CDB-DE40478775C0@gmail.com> Hi im anibal so i was looking for a method faster than miniprep or colony pcr and i found this one so i just made it but no get succesfull, please will you send me your protocol detailed with t?ps and any advice? Enviado desde mi iPod From mgbiotec from gmail.com Tue Oct 27 22:18:33 2009 From: mgbiotec from gmail.com (megha goyal) Date: Wed Oct 28 10:36:32 2009 Subject: Inclusion bodies centrifugation Message-ID: Dear All, Our protein is expressed as inclusion bodies and I want to separate inclusion bodies from E.coli from the cellular debris after* *lysis of the cells by sonication. Can I do this by normal centrifugation? and if yes, at what speed? Our centrifuge has maximum speed of 14000 rpm. Can we do the separation using this centrifuge and if so how. Thanking in anticipation. From mgbiotec from gmail.com Tue Oct 27 23:16:59 2009 From: mgbiotec from gmail.com (megha goyal) Date: Wed Oct 28 10:36:39 2009 Subject: Inclusion bodies centrifugation In-Reply-To: References: Message-ID: Thanks for the reply. actually we do not use any lysis buffer just suspend our e.coli cells in water and perform sonication thereafter we centrifuge at 140000 rpm to collect the inclusion bodies and these are contaminated with host cell proteins too. as mentioned by artem, i just wanted to know if we centrifuge at 5000 - 6000 rpm then will the cell debris not settle down. kindly guide. as such i shall try the method and revert back. thanks for the reply On 10/27/09, megha goyal wrote: > > Dear All, > > Our protein is expressed as inclusion bodies and I want to separate > inclusion bodies from E.coli from the cellular debris after* *lysis of the > cells by sonication. > > Can I do this by normal centrifugation? and if yes, at what speed? > > Our centrifuge has maximum speed of 14000 rpm. Can we do the separation > using this centrifuge and if so how. > > > > Thanking in anticipation. > From cathalgarvey from gmail.com Wed Oct 28 06:51:51 2009 From: cathalgarvey from gmail.com (Cathal Garvey) Date: Wed Oct 28 10:36:45 2009 Subject: adding kozak sequence to primer In-Reply-To: <615744.83799.qm@web52705.mail.re2.yahoo.com> References: <615744.83799.qm@web52705.mail.re2.yahoo.com> Message-ID: <468b1a400910280451h8fd1abaj98d538b4713c9568@mail.gmail.com> Using the Kozak you have is just fine, I'd say. It's a "Consensus" sequence, which means the standard Kozak you find online is the average of hundreds or thousands of sequenced Kozaks from natural sequences. Slight deviations from the consensus are the norm, and shouldn't have a huge impact. In fact, it may be an important part of the gene's natural regulation. If you have the time and resources to replace the nucleotide and bring it to full consensus, it might be worth it just to have the sequence on hand in the future. And it might look better on a paper, though personally I wouldn't nitpick such small details if I were reviewing it. From rwmd1 from NOSPAMle.ac.uk Wed Oct 28 02:49:11 2009 From: rwmd1 from NOSPAMle.ac.uk (Raymond Dalgleish) Date: Wed Oct 28 10:36:54 2009 Subject: Loading dye In-Reply-To: References: <200910261704.n9QH46M03272@net.bio.net> <7knstaF3akt33U1@mid.individual.net> Message-ID: The issue is now solved. We ran a 1% agarose gel of the suspect loading dye alongside size markers and other loading dyes of known provenance. The dye which had been described as being bromophenol blue was in fact very highly concentrated xylene cyanol, so it looked dark blue rather than blue/green. The person responsible will be re-educated. Thanks for all the replies. Raymond Jayakumar, R wrote: > > Hmm.. XC I think moves with the 4K band and above the blue dye front of BPB (the blue dye he is talking about) moves with a 300 bp band on a 1% gel > Jay > > > -----Original Message----- > From: methods-bounces@oat.bio.indiana.edu [mailto:methods-bounces@oat.bio.indiana.edu] On Behalf Of Christian Praetorius > Sent: Tuesday, October 27, 2009 5:30 AM > To: methods@magpie.bio.indiana.edu > Subject: Re: Loading dye > > Barbara MacGregor wrote: > >> Xylene cyanol definitely has a different color, though, at least as >> I've used it - blue-green rather than blue. > > Thats right. But it migrates around 10k in a 1% gel. > > Christian > > -- > X-no-Sig: yes > _______________________________________________ > Methods mailing list > Methods@net.bio.net > http://www.bio.net/biomail/listinfo/methods > > > This email message may contain legally privileged and/or confidential information. If you are not the intended recipient(s), or the employee or agent responsible for the delivery of this message to the intended recipient(s), you are hereby notified that any disclosure, copying, distribution, or use of this email message is prohibited. If you have received this message in error, please notify the sender immediately by e-mail and delete this email message from your computer. Thank you. From novalidaddress from nurfuerspam.de Wed Oct 28 11:04:00 2009 From: novalidaddress from nurfuerspam.de (WS) Date: Wed Oct 28 12:03:51 2009 Subject: Loading dye References: <200910261704.n9QH46M03272@net.bio.net> <7knstaF3akt33U1@mid.individual.net> Message-ID: > The person responsible will be re-educated. Means hopefully: Will be sentenced to bake Black-Forest-Cake (under surveillance) for the next lab meeting? Cheers, Wo From pjie2 from cam.ac.uk Wed Oct 28 11:10:54 2009 From: pjie2 from cam.ac.uk (Peter Ellis) Date: Wed Oct 28 12:03:56 2009 Subject: adding kozak sequence to primer In-Reply-To: References: <615744.83799.qm@web52705.mail.re2.yahoo.com> Message-ID: <7kr8oiF3b6v14U1@mid.individual.net> Cathal Garvey wrote: > Using the Kozak you have is just fine, I'd say. It's a "Consensus" sequence, > which means the standard Kozak you find online is the average of hundreds or > thousands of sequenced Kozaks from natural sequences. Slight deviations from > the consensus are the norm, and shouldn't have a huge impact. In fact, it > may be an important part of the gene's natural regulation. > > If you have the time and resources to replace the nucleotide and bring it to > full consensus, it might be worth it just to have the sequence on hand in > the future. And it might look better on a paper, though personally I > wouldn't nitpick such small details if I were reviewing it. I think it would be a very bad idea to change it. It's a non-synonymous substitution, which means you have a non-zero risk of altering your protein's function. Meanwhile, one presumes that the native gene gets translated fine despite not being a perfect match to the consensus. So why change it? Peter From fromsolar from foxmail.com Wed Oct 28 21:42:23 2009 From: fromsolar from foxmail.com (=?gbk?B?ZnJvbXNvbGFy?=) Date: Wed Oct 28 22:09:30 2009 Subject: something about AflII Message-ID: RGVhciBKb2huo6wNCiAgICAgSSBhbSBhIHBvc3QtZ3JhZHVhdGUgaW4gZnVqaWFuIG5vcm1h bCB1bml2ZXJzaXR5IGluIGNoaW5hLkkgaGFwcGVuIHRvIHNlZSBhIGxldHRlciB5b3Ugd3Jv dGUgYWJvdXQgQWZsSUksSSBoYXZlIHNvbWUgcHJvYmxlbSBpbiBsaWdhdGUsY2FuIHlvdSB0 ZWxsIG1lIHdoYXQgc2hvdWxkIEkgbm90aWNlIGluIHRoZSBwcm9jZXNzIG9mIGxpZ2F0ZS4N CiAgDQogU2luY2VyZWx5IHlvdXJzLA0KICANCiANCiANCldhbmxpbmcgQ2FpDQogDQogDQog DQpFbmdpbmVlcmluZyBSZXNlYXJjaCBDZW50ZXIgb2YgSW5kdXN0cmlhbCBNaWNyb2Jpb2xv Z3kgb2YgTWluaXN0cnkgb2YgRWR1Y2F0aW9uLCBhbmQgQ29sbGVnZSBvZiBMaWZlIFNjaWVu Y2VzLCBGdWppYW4gTm9ybWFsIFVuaXZlcnNpdHksIEZ1emhvdSAzNTAxMDgsIFAuIFIuIENo aW5hIA0KIA0KUGhvbmU6IDg2LTU5MS0yMjg2ODIxMiANCiANCkZheDogODYtNTkxLTIyODY4 MTk1DQogDQpFLW1haWw6ZnJvbXNvbGFyQGZveG1haWwuY29t From shifalich from rediffmail.com Thu Oct 29 02:53:38 2009 From: shifalich from rediffmail.com (shifali chatrath) Date: Thu Oct 29 13:46:13 2009 Subject: how to prevent aggregation Message-ID: <1256679829.S.5503.51990.f4mail-235-211.rediffmail.com.1256802818.23481@webmail.rediffmail.com> Dear all! I have express soluble protein in origami with pET 32a vector that has Trx (Thioredoxin) tag. But, the problem is aggregation. They are soluble aggregates. In spite of adding NaCl, DTT, EDTA Glycerol etc.( these are the things which are recommended in few papers to overcome aggregation), its still showing aggregation. I cant crystallize the protein or do NMR unless I get rid of this problem. Inspite of aggregation, the protein is active, but probably, it will be more potent if it is in soluble aggregate free form. I seek advice from all of you over this. Please suggest any possible way out or share your own practical experiences over this. Thanks REgards Shifali Shifali Chatrath Graduate Student Protein science Lab Dept. of Biological sciences National University of Singapore Singapore +65-65161210 +65-96393449 From holeung.ng from gmail.com Fri Oct 30 13:41:25 2009 From: holeung.ng from gmail.com (Ho Leung Ng) Date: Fri Oct 30 15:09:25 2009 Subject: how to prevent aggregation Message-ID: <6678762a0910301141r49f69588ude1ea9aeb3aa3097@mail.gmail.com> Have you tried expressing at lower temperature or with much less IPTG? Try adding ligand/inhibitor with your protein, changing pH, or adding detergent. The Trx tag may be helping to solubilize your aggregated protein. If you cut it off, your protein may crash out of solution. If it's a small protein, you could try refolding.